Hive-mounted disseminator device

ABSTRACT

The present invention relates to the dissemination of biological control agents or other substances through the use of bees, in particular bumblebees. It relates to a disseminator device, installable in or in connection to the hive, and which contains biological control agents or other substances which are picked up, carried and disseminated by the bees when leaving the hive.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/321,348, filed Nov. 18, 2011, which is a national-stage entry ofInternational Application PCT/EP2010/057508, filed May 29, 2010, whichinternational application claims the benefit of priority to GreatBritain Patent Application No. 1006909.4, filed Apr. 26, 2010, and toGreat Britain Patent Application No. 0909172.9, filed May 29, 2009.

FIELD OF THE INVENTION

The present invention relates to the dissemination of biological controlagents or other substances through the use of bees, including honeybeesand bumblebees, with in particular bumblebees. It relates to adisseminator device, installable in or in connection to the hive, andwhich contains biological agents or other substances includingagrochemicals, which are picked up, carried and disseminated by the beeswhen foraging outside the hive.

BACKGROUND TO THE INVENTION

Crops need to be protected against a broad range of diseases and pests.Flowers can be important pathways of plant disease infection or sites ofpest occurrence. The pathogen infects them under favorable conditions,and progressively colonizes other tissues or fruits. Disease symptomsbecome visible when infected tissues ripen, senesce or die, as forexample in the case of Botrytis cinerea. Effective disease and pestcontrol depends both on the use of suitable crop protection products,either chemical or biological (microbial or microbial agents), and onthe methods and strategies for introducing, promoting and maintainingthe antagonist in the crop (Sutton and Peng, Annual Review ofPhytopathology, 1993, 31, pg. 473-493).

The use of microbiological antagonist preparations against flower-bornediseases and pests could be particularly effective if dissemination ofthe antagonist occurs directly to the flower parts during bloom (Pengand Sutton, Canadian Journal of Plant Pathology, 1991, 13, pg. 247-257).Pronubial insects are known to deliver pollen, fungi and bacteria (Daget al., Journal of Apicultural Research, 2000, 39, Pg. 117-123). Hence,while performing their pollination service, pronubial insects might alsoserve as carriers of antagonistic agents, providing potential diseaseand pest control in addition to pollination services (Sutton and Peng,Annual Review of Phytopathology, 1993, 31, pg. 473-493; Sutton, Advancesin Plant Pathology, 1995, 11, 171-188).

Worldwide over 500,000 colonies of B. terrestris (L.) are purchasedannually for pollination service on various crops, predominantly ontomatoes. In addition to pollination and indirect disease control, thesepronubial insects may also perform an active role in preventing diseaseor pest control by conveying for example microbiological preparationsdirectly onto flowers or other plant parts. This requires that the beesthemselves are previously loaded with propagules of the antagonist(Sutton and Peng, Annual Review of Phytopathology, 1993, 31, pg.473-493). For the latter a dispenser can be mounted on the exit of thebeehive. Existing dispenser such as the side-by-side passagewaydispenser (SSP) and the overlapping passageway dispenser (OP) havecertain drawbacks in loading the bees.

The SSP-dispenser showed several functional limits, as evidenced by theabsence of antagonistic propagules on most of the captured and analyzedbees. Inoculum density on bumblebees was also rather low. TheSSP-dispenser was also not efficient in separating outgoing and incomingbees: many bees exited the colony box through the darkened straightpassageway thus eluding the powder preparation, whereas others crawledthrough the zigzag passageway, but walked along the side walls.Furthermore the bees promptly smeared the zigzag passageway with theirliquid excrements which, once kneaded with the antagonistic powderpreparation, caused the latter to lose its consistency making it nolonger suitable for bee loading (Maccagnani B. et al., 2005, Bulletin ofInsectology 58(1): 3-8)

With the OP dispenser only a limited amount of propagules of theantagonist can be loaded in the lower exit passage and the loading ofthe bees leaving the hives quickly diminishes. A further disadvantage ofthe OP-dispenser is the different dimensions of the upper and lowerpassageway. The upper passageway is broader and higher compared to thelower passageway, as a consequence and given the range in size ofbumblebees in a hive, some of the larger bees may leave the hive throughthe upper passageway of the OP-dispenser and get stuck in the narrowerlower passageway. Also, the difference in floor level between the upperand the lower passageway has a negative effect on light entering theexit hole of the hive.

In addition, for both the SSP-dispenser and the OP-dispenser, theinoculum density on flowers and the percentage of colonized flowers weresignificantly lower than in comparable spray treatment (Maccagnani B. etal., 2005, Bulletin of Insectology 58(1): 3-8). Also, both the SSP andOP dispenser are at the exterior of the beehive, and accordinglysensitive to changes in temperature. For example fully exposed in agreenhouse the temperature increase within the dispenser may not only bedetrimental to the control agent but also prevents the bees from leavingthe hive.

It is accordingly a general object of the invention to provide a beehivedispenser to overcome the problems mentioned hereinbefore. As providedin more detail hereinafter, the beehive disseminator of the presentinvention assures a unidirectional exit passage of the bees through thedispenser, thereby preventing bees from entering through the exit (i.e.from entering through the dispenser). An independent and againunidirectional entry pathway is realized through a second chamber,thereby preventing bees from leaving the hive through the entrance. In aparticular embodiment the exit and entrance are integrated into onevisual unit, contributing to a swift entry by returning bees.Furthermore, the dispenser has a high loading capacity for the agent tobe disseminated, it prevents that the bees clear a path in the product,which would prevent effective loading, and thus provides an optimalloading of the bees with a short passage time. The dispenser can befilled through perforation in the top of the chamber. Finally, it can beintegrated within a hive to benefit from the internal temperaturecontrol. It can be made accessible through a removable top, a slidingmechanism or other means.

SUMMARY OF THE INVENTION

A hive disseminator device comprising a chamber (2); said chamber havingapertures (4,5) at opposite side members of said chamber and havingmeans (8) to amass and immobilize a substance at the floor member ofsaid chamber; and characterized in that the apertures at the oppositeside members of said chamber include means (5) to define aunidirectional pathway for bees through said chamber.

In a further embodiment, the hive disseminator further comprises asecond, separate chamber (1) wherein said second chamber has apertures(3,6) at opposite side members and said apertures include means (3) todefine a unidirectional pathways for bees through said second chamber.In a particular embodiment the unidirectional pathway through saidsecond chamber is opposite to the unidirectional pathway through thechamber having means to immobilize a substance at its floor member,hereinafter also referred to as the dispenser chamber (2).

In said dispenser chamber (2) the means (8) to amass and immobilize asubstance at the centre of the floor member, hereinafter generallyreferred to as means to immobilize a substance at the floor member ofsaid chamber, are such that they prevent, either alone or incombination, that the bees are able to clear a path through thesubstance to be disseminated and are for example selected from one ormore of a plurality of edges, a mesh, a series of slopes separated by ashallow edge, a halfpipe, ribbed paper, or the like. In a particularembodiment the means to immobilize a substance consists of a mesh (8),or a plurality of edges, ribs, ridges, bumps, etc. In a more particularembodiment the means to immobilize the substance at the floor memberconsists of a series of slopes separated by a shallow edge, said seriesof slopes being an integrated part of the floor member; or of a halfpipewith its longitudinal axis in the direction of the unidirectionalpathway through said chamber, optionally comprising two or more ofedges, ribs, ridges, bumps, etc. The means to immobilize the substanceat the floor member can either be removable or are an integrated part ofthe floor member of the dispenser chamber. In a particular embodimentsaid means to immobilize a substance at the floor member of said chamberare an integrated part of said floor member. In a further embodimentsaid means are removable.

In both of the aforementioned chambers, the means to define aunidirectional pathway through said chamber consist of one or moredownward directed exits at one end of said chamber and one or moreentrances at the opposite end of said chamber. Alternatively, the meansto define the unidirectional pathway may consist of a one-way door (9),such as for example a swing door, a trapdoor or hatch that opens in asingle direction. In a particular embodiment the one way-door maycomprise a small opening, e.g. at its base. Said opening lowers theinitial barrier and encourages the bees to open the door.

As exemplified, the means to define a unidirectional pathway typicallyconsist of outwardly directed and diagonally cut tubes or one-way doors,in particular diagonally cut conical tubes (3,5) or swing doors (9) andthe entrances typically include means (4,3) to communicate with a hiveentrance or exit. The means to communicate with a hive entrance or exitare usually selected from flexible tubes, rigid tubes, one-way doors orconical tubes. In an even further embodiment the entrances can beclosed, such as for example with a sliding. In case the means to definea unidirectional pathway is part of an actual exit from the hive, itpreferably consists of a transparent material, more preferably atranslucent material, i.e. such as a transparent or translucentdiagonally cut conical tube, or a transparent or perforated one-waydoor. In case the means to define a unidirectional pathway is part of anactual entrance to the hive, it preferably consists of an opaquematerial, i.e. such as an opaque (dark) diagonally cut conical tube,alternatively an opaque one-way door.

In a particular embodiment of the hive disseminator device of thepresent invention, the chamber having the means to immobilize asubstance, i.e. the dispenser chamber (2), defines an exit pathway forbees leaving the hive; and the second chamber (1) defines a pathway forbees entering the hive. In said embodiment the exit pathway is at least5, 10, 15, 20 or 25 cm long.

In one embodiment of the present invention the chamber having the meansto immobilize a substance, i.e. the dispenser chamber (2) has arectangular base; one, two or more means to define a unidirectionalpathway consisting of outwardly directed and diagonally cut transparentconical tubes (5) or one-way doors (9); a tetragonal entrance (4) and isfurther characterized in having an edge proximal to the entrance, toretain the means to immobilize a substance at the floor member of saidchamber. In a particular embodiment said two or more means to define aunidirectional pathway through the dispenser chamber are at oppositeside members of the dispenser chamber.

In a further aspect of said embodiment, the second chamber (1) has arectangular base; a unidirectional exit consisting of an outwardlydirected and diagonally cut opaque conical tube (3) or an opaque one-waydoor (9); and an entrance (6) optionally including means thatcommunicate with the hive entrance, such as for example diagonally cuttransparent conical tubes (5) or one-way doors (9).

In an alternative embodiment of the present invention the chamber havingthe means to immobilize a substance, i.e. the dispenser chamber has atrapezoid base and is further characterized in that the aperture(s) atthe short side wall of said trapezoid base include means (4) tocommunicate with a hive exit. The long side wall of said trapezoid baseddispenser chamber has one, two or more downward directed exitsconsisting of outwardly directed and diagonally cut transparent conicaltubes (5).

In a further aspect of said embodiment the second chamber (1) has atrapezoid base; a downward directed exit consisting of an outwardlydirected opaque conical tube (3); and an entrance (6) including meansthat communicate with the hive entrance.

The two chambers making up the hive disseminator devices as describedherein could consist of two separate chambers, but as exemplified, in aparticular embodiment the chamber having the means to immobilize asubstance, i.e. the dispenser chamber; and the second chamber takentogether, define a single housing. In said particular embodiment the twochambers are either adjacent or superimposed, with in a more particularembodiment the dispenser chamber on top of the second chamber andfurther characterized in that the exit (5) of the dispenser chamber issuperimposed vis-à-vis the entrance (6) of the second chamber. Thesuperimposed exit (5) and entrance (6) are optionally framed with avisual mark (10), in particular a blue mark.

As such, the hive disseminator devices as defined herein can be placedoutside and connected to the hive entrance and exit, or it can be placedwithin the hive. When placed inside the hive, the hive disseminatordevices as defined herein will include means facilitating mounting ofthe device in a hive. In a particular embodiment the hive disseminatordevice is an integrated part of the hive top grid.

It is accordingly also an object of the present invention to provide ahive comprising a disseminator device as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of the different embodiments of the presentinvention only, and are presented in the cause of providing what isbelieved to be the most useful and readily description of the principlesand conceptual aspects of the invention. In this regard no attempt ismade to show structural details of the invention in more detail than isnecessary for a fundamental understanding of the invention, thedescription taken with the drawings making apparent to those skilled inthe art how the several forms of the invention may be embodied inpractice.

FIG. 1A: Funnel-dispenser, rear view, 1: entrance compartment, 2:trapezium-shaped exit compartment, 3: bumblebee in-closer connecting theentrance compartment with the bee hive, 4: connection of the exitcompartment with the bee hive, 5: bumblebee in-closer exit openings, 6:entrance opening, 7: lid of the dispenser.

FIG. 1B: Funnel-dispenser, side view, 1: entrance compartment, 2:trapezium-shaped exit compartment, 3: bumblebee in-closer connecting theentrance compartment with the bee hive, 4: connection of the exitcompartment with the bee hive, 5: bumblebee in-closer exit openings, 6:entrance opening, 7: lid of the dispenser.

FIG. 1C: Funnel-dispenser, front view, 1: entrance compartment, 2:trapezium-shaped exit compartment, 3: bumblebee in-closer connecting theentrance compartment with the bee hive, 4: connection of the exitcompartment with the bee hive, 5: bumblebee in-closer exit openings, 6:entrance opening, 7: lid of the dispenser.

FIG. 1D: Funnel-dispenser, top view, 1: entrance compartment, 2:trapezium-shaped exit compartment, 3: bumblebee in-closer connecting theentrance compartment with the bee hive, 4: connection of the exitcompartment with the bee hive, 5: bumblebee in-closer exit openings, 6:entrance opening, 7: lid of the dispenser.

FIG. 2: Impact of the length of the Funnel-dispenser on the efficiencyof loading of the body of workers of B. terrestris. The average numbersof CFU/bee are expressed as means (±SEM). ANOVA resulted in two groups(F=29.981; DF=149; P<0.001). Values that are followed by a differentletter (a-b) are significantly different (Tukey-Kramer post-hoc testwith P=0.05).

FIG. 3: Impact of the Funnel-dispenser system, when placed before thenest, on bumblebee foraging activity at different time points, beforeand after loading of the dispenser.

FIG. 4: Comparison of the F-dispenser with the modified SSP-dispenser ina closed greenhouse on the amount of deposition of MCA on flowers. Meannumber of CFU's recovered per zone (A-B-C) per type of dispenser[modified SSP-dispenser (black bars) or F-dispenser (grey bars)] duringa three-week sampling, with samples taken once a week.

FIG. 5A: Rectangular-dispenser, built-in in a bumblebee hive, top viewwith closed lid, 1: entrance compartment, 2: rectangular-shaped exitcompartment, 3: bumblebee in-closer connecting the entrance compartmentwith the bee hive, 4: connection of the exit compartment with the beehive, 5: bumblebee in-closer exit openings, 6: entrance opening, 7: lidof the dispenser.

FIG. 5B: Rectangular-dispenser, built-in in a bumblebee hive, top viewwith open lid, 1: entrance compartment, 2: rectangular-shaped exitcompartment, 3: bumblebee in-closer connecting the entrance compartmentwith the bee hive, 4: connection of the exit compartment with the beehive, 5: bumblebee in-closer exit openings, 6: entrance opening, 7: lidof the dispenser.

FIG. 5C: Rectangular-dispenser, built-in in a bumblebee hive, side view,1: entrance compartment, 2: rectangular-shaped exit compartment, 3:bumblebee in-closer connecting the entrance compartment with the beehive, 4: connection of the exit compartment with the bee hive, 5:bumblebee in-closer exit openings, 6: entrance opening, 7: lid of thedispenser.

FIG. 5D: Rectangular-dispenser, built-in in a bumblebee hive, frontview, 1: entrance compartment, 2: rectangular-shaped exit compartment,3: bumblebee in-closer connecting the entrance compartment with the beehive, 4: connection of the exit compartment with the bee hive, 5:bumblebee in-closer exit openings, 6: entrance opening, 7: lid of thedispenser.

FIG. 6: Comparison of the R-dispenser and the SSP-dispenser on foragingactivity of bumblebees. Mean foraging activity per 30 min was measuredat 3 consecutive days and 2 different time points per day for 3 beehiveswithout dispenser (control), 3 beehives with the R-dispenser and 3beehives with an S-dispenser.

FIG. 7: Effect of loading of the R-dispenser on foraging activity ofbumblebees. Mean foraging activity per 30 min measured at 3 differenttime points (7 AM, 11 AM and 4 PM) the day after loading of theR-dispenser. Loaded R-dispenser (black bars), empty R-dispenser (greybars).

FIG. 8: R-dispenser, built into a in a bumblebee hive, showing bumblebeein-closer exit openings (5) having a one-way door (9), and an entranceopening framed with a visual mark (10) (i.e. a blue mark).

FIG. 9A: Superimposed Dispenser (S-Dispenser) Top Front View, built intoa in a bumblebee hive, showing the superimposed exit (5) and entrance(6) framed with a visual mark (10).

FIG. 9B: Superimposed Dispenser (S-Dispenser) Bottom Rear View, builtinto a in a bumblebee hive, showing the superimposed exit (5) andentrance (6) framed with a visual mark (10).

FIG. 9C: Superimposed Dispenser (S-Dispenser) Side View, built into a ina bumblebee hive, showing the superimposed exit (5) and entrance (6)framed with a visual mark (10).

FIG. 9D: Superimposed Dispenser (S-Dispenser) S-Dispenser, built into ain a bumblebee hive, showing the superimposed exit (5) and entrance (6)framed with a visual mark (10).

FIG. 9E: Superimposed Dispenser (S-Dispenser), representation of apossible embodiment of the means (8) to immobilize the substance to bedisseminated in the form of a half pipe having a plurality of ridges.

FIG. 10: 3 valves S-dispenser, a modified S-dispenser featuring a thirdvalve between the dispenser chamber and the nest.

FIG. 11: Results of Example 3, showing mean foraging activity per 30minutes for each dispenser type.

DETAILED DESCRIPTION OF THE INVENTION

The disseminator device of the present invention solves the problemsassociated with the aforementioned prior art devices in that itprovides:

-   -   a high loading capacity for the agent to be disseminated,    -   it provides an optimal loading of the bees with a short passage        time,    -   it has no influence on the foraging behavior of the bees,    -   it can be integrated within a hive, and    -   it is easily accessible for refilling with a minimal disturbance        of the nest.

In order to achieve the aforementioned objectives the beehivedisseminators of the present inventions, in the examples hereinafteralso referred to as dispensers, comprise a chamber to receive thebiological control agents or other substances to be disseminated by thebees. As for example shown in the figures, the dispenser chamber iseasily accessible for refilling purposes. In order to prevent bees fromleaving the hive whilst manipulating the dispenser chamber, the entrance(4) to said chamber is preferably accommodated with means to close theentrance, such as for example a sliding door. Accessibility to thedispenser chamber could for example be realized by means of a removablelid (7), optionally hinged connected to the top grid (0) of the hive.Alternatively, and in case the beehive disseminator of the presentinvention is integrated into the hive, the top member of the dispenserchamber may consist of the covering grid (0) of the hive. In saidinstance refilling of the dispenser chamber can be achieved directlythrough the grid.

As already mentioned hereinbefore, the beehive disseminators of thepresent invention are characterized in that they comprise one or moreentrances and exits that taken together define a unidirectional pathwayfor the bees through said chamber. When used in conjunction with a hive,this unidirectional pathway is either an entrance pathway for beesentering the hive or an exit pathway for bees leaving the hive. In apreferred embodiment, the unidirectional pathway through the chamberreceiving the substance to be disseminated, hereinafter also referred toas the dispenser chamber, is part of the exit pathway for bees leavingthe hive.

To realize the unidirectional pathway through the chamber the exitsshould be such that they allow bees to leave the chamber through thecorresponding opening, but prevents bees from entering through said exitopening. As it is known that bees will not enter the hive through adownward directed hole, the means to define a unidirectional pathwaythrough said chamber may consist of one or more downward directed exitsat one end of said chamber and one or more entrances at the opposite endof said chamber. The downward directed exit could for example berealized with a cap or hood that fits externally over the exit openingin the side member of said chamber and which has an opening in thedownward right-angled plane when compared with the exit opening in theside member of said chamber. Alternatively, the downward directed exitcould be realized with a diagonally cut conical tube that fits in andextends to the exterior of the exit opening. The downward directed exitscould be made of any material typically used in the manufacture of beehive dispensers, but in case the exit is actually part of the exitpathway for bees leaving the hive, the material should be lighttransparent as departing bees are responsive to light entering thechamber.

In an even further alternative embodiment, the means to realize aunidirectional pathway could be realized with a one-way door, such as atrapdoor, swing door or hatch. To encourage the bees in opening thedoor, and as shown in the examples hereinafter, in a particularembodiment the one-way doors comprise at their base a small opening.There are no particular requirements to the shape of said opening, butits size should be such that it allows bees to identify the opening as apossible escape route, but small enough to prevent them from gettingthrough. As such the width and/or height of this small opening is fromabout 2 mm to about 3 mm. Again, in case said one-way door is part ofthe exit pathway, the door should be made of a light transparent,preferable light translucent or perforated material. In case the one-waydoor is part of an actual entrance to the hive, it preferably consistsof an opaque (dark) material. In the latter and to encourage the bees inopening the door, said door may comprise a further opening, such as forexample at the base of said door enabling the scent of the hive to enterthe second chamber (1).

Different from the art known dispensers, the dispenser chamber ofpresent beehive disseminators is substantially free of obstacles likethe cross plates found in the SSP dispenser and the floor level changesfound in for example the OP dispenser. The latter influence the passagetime in the dispenser, the infiltration of light in the hive andaccordingly are likely to influence the foraging behavior of the bees.Compared to the aforementioned disseminators, the dispenser chamber ofthe present invention only contains means at the floor member of saidchamber to immobilize the substance to be disseminated, but withouthindering the passage of the bees when walking through the dispenserchamber. As already explained hereinbefore, said means prevent that thebees are able to clear a path through the substance to be disseminated.

The substance to be disseminated can be any substance that is notpathogenic to the bees, including pollen; plant protection products suchas agrochemicals; biocontrol agents such as for example virus, bacteria,fungi, rickettsia, nematode, eggs of beneficial mites or insects, orbeneficial mites or insects; fertilizers; or preparations containingplant hormones, etc. While it is well known in the art that bumblebeehives without dispenser and disseminated substance included therein areuseful for pollination of certain plants, the disseminated substance asused in the invention is intended to deliver an added utility to theplant (preferably via the flowers) visited by the bumblebees such asreducing certain pest and disease problems, pollination (in particularcross-pollination) or better or altered development of the flower or theplant.

Said substances can be used as such, or in the form of a formulation,including combinations of the aforementioned substances. In a particularembodiment, the substance is in a liquid or powdered formulated, more inparticular the formulation is a powdered formulation, e.g. using apowdered carrier. The powdered carrier can be a starch such as corn,starch, talc, dust derived from crushed hulls of nuts, clay dust, ormixtures thereof.

Any material to prevent unwanted displacement of the powderedsubstance/formulation can be used in the dispenser chamber of thepresent invention. In a particular embodiment said means are anintegrated part of the floor member of the dispenser chamber, forexample in case an abrasive material is used to make the floor member ofsaid chamber. Examples of such an abrasive material include rough-cutwood that is naturally textured or plastic having a non-skid surface,e.g. having a scribed surface or a plurality of ridges. In an evenfurther embodiment said integrated means to prevent the unwanteddisplacement of the substance consists of a mesh, or a plurality ofedges, ribs, ridges, bumps, etc. In a particular embodiment theintegrated means to prevent the unwanted displacement of the substanceconsist of a series of slopes separated by a shallow edge. Theinclination of said slopes is preferably in the same directional axis asthe exit pathway through said chamber. In said particular embodiment thebees will dip a number of times through the substance to bedisseminated, resulting in a high load over a short distance.Alternatively, the floor member of the dispenser chamber is covered witha removable material, and are for example selected from one or more of amesh screen, e.g. of welded wire; cheesecloth; sponge; brushes; ribbedpaper; a series of slopes separated by a shallow edge, a halfpipe, orribbed paper. In a particular embodiment said means are removable andmay consist of a combination of any one of the aforementioned examples.In a preferred embodiment the means to immobilize the substance to bedisseminated consists of a removable halfpipe comprising two or more ofedges, ribs, ridges, bumps, etc. (such as for example represented inFIG. 9E). Blocking members to retain said material at a fixed positionin the dispenser chamber may also be present. In any one of theaforementioned embodiments, the means to prevent the unwanteddisplacement of the substance will cause an automatic and evendistribution of the substance, even after a bee crossed the dispenserchamber. For example, in case the means to prevent unwanted displacementof the substance consists of ribbed paper, the ribs are preferentiallyin the longitudinal direction of the dispenser chamber.

As provided in more detail in the examples hereinafter, in a particularembodiment the beehive disseminator of the present invention is builtinto an actual beehive. One of said integrated beehive disseminatorswill consist of a dispenser chamber as described hereinbefore, whereinthe unidirectional exit(s) of the dispenser chamber communicate with theexit(s) of the hive and the entrance(s) of the dispenser chamber is indirect contact with the interior of the hive. To improve theaccessibility of the integrated dispenser chamber for bees willing toleave the hive, the hive optionally comprises means that allow bees tocrawl from the hive up and into the dispenser chamber, such as forexample a wire, stick, mesh, ladder, etc . . . , that extends from theentrance (4) of the dispenser chamber into the hive. It is accordinglyan object of the present invention to provide a beehive disseminator ofthe present invention further characterized in that the dispenserchamber comprises at its entrance (4) means that allow bees to crawlfrom the hive up and into the dispenser chamber. Said means that allowbees to crawl from the hive up and into the dispenser chamber, are forexample attached to the dispenser chamber, or to the top grid of thehive.

As already mentioned hereinbefore, since bees looking to exit areresponsive to light entering the hive, it is of importance thatsufficient light enters the dispenser chamber. Further to the use oftransparent or perforated downwardly directed exit means (supra), thelight in the dispenser chamber can be enhanced through the use of atransparent or perforated side member at the exit of the dispenserchamber. In said embodiment wherein the beehive disseminator of thepresent invention is built into an actual beehive, the transparent orperforated side member at the exit of the dispenser chamber shouldeither be in contact with a corresponding window or opening in theactual beehive. It is accordingly an object of the present invention toprovide a beehive disseminator of the present invention furthercharacterized in that the dispenser chamber comprises a transparent orperforated side member at the exit of said chamber.

To assure that bees leaving the hive will pass through the dispenserchamber and not escape via the beehive entrance, the latter shouldcomprise similar means as the means used for the unidirectional exitsmentioned above. In a particular embodiment the hive entrance hole willcomprise an inwardly directed conical tube, optionally a diagonally cutconical tube. To prevent light penetration, which may attract departingbees, the means to realize the unidirectional entrance should be made ofa light opaque material. In an even further alternative embodiment, thebeehive entrance could be realized with a one-way door, such as atrapdoor, swing door or hatch. As for the diagonally cut conical tubeabove, the one-way door at the entrance to the hive, is preferably madeof a light opaque material.

Where bees leaving the hive are responsive to light, bees entering thehive are attracted by the scent of the hive. Thus in case the means torealize the unidirectional entrance consist of a one-way door, thelatter may comprise a further opening, such as for example at the baseof said door enabling the scent of the hive to leave and attract theentering bees.

Thus in a particular embodiment, the present invention provides a hivecomprising a dispenser chamber as described hereinbefore, wherein;

-   -   the unidirectional exit(s) of said chamber communicate with the        hive exit(s);    -   the entrance(s) of said chamber are in direct contact with the        interior of the hive; and        wherein the hive entrance comprises means to realize a        unidirectional entrance.

As already mentioned hereinbefore, the means to realize a unidirectionalentrance could be similar to the means to realize the unidirectionalexit, but alternatively the hive entrance will communicate with theentrance of a second chamber. As for the dispenser chamber, the exitopening(s) in said second chamber should be such that taken togetherwith the entrance they define a unidirectional pathway for the beesthrough said chamber. When integrated in the hive, the exit opening ofsaid second chamber is in direct contact with the interior of the hiveand comprises means to prevent bees from entering said second chamberthrough said exit opening. In a particular embodiment, said meansconsist of an inwardly directed conical tube, optionally a diagonallycut conical tube. In an alternative embodiment said means consist of aone-way door, such as a trapdoor, swing door or hatch that opens in asingle direction. In an even further embodiment the integrated entrancechamber (the second chamber (1)) may comprise at its unidirectional exitthat is in direct communication the interior of the hive, means thatallow bees to crawl from the entrance chamber down and into the hive,such as for example a wire, stick, mesh, ladder, etc . . . .

Where the length of the exit pathway is important to realize an optimalload of the bees with the substance to be disseminated, the length ofthe entrance pathway is of less importance. Consequently, compared tothe length of the dispenser chamber, the second chamber may have thesame length but could be shorter as well. To further reduce lightpenetration through the hive entrance pathway, the length of the secondchamber should be about 5, 10, 15 cm or more. To benefit from the scentof the hive in attracting entering bees, the second chamber could bemade of a mesh, or comprise perforations at its base and side member(s)in communication with the hive. To allow the development of a scenttrail in the second chamber, the latter may comprise a carrier materialcovering the base (floor) of said chamber, such as for example a pieceof cardboard, or rough-cut wood. Again and similar to the unidirectionalentrance mentioned hereinbefore, in case the unidirectional exit of saidsecond chamber consists of a one-way door, the latter may comprise afurther opening, such as for example at the base of said door enablingthe scent of the hive to enter said chamber and attract the enteringbees.

Again, but for the length of the exit pathway, there are no particularlimitations to the shape of the chambers as shown in the examples hereinbelow. Given the ability to integrate the chambers into a hive,limitations in the height, width and length are typically set by thedimensions of the hive. Width of the chambers is typically from about2.0 to 15.0 cm, with the dispenser chamber on average from about 5.0 toabout 15.0 cm and the second chamber on average from about 2.0 to about5.0 cm. The height of the chambers should be such that it prevents beesfrom flying and is typically from about 1.0 to about 15.0 cm with onaverage a height of about 3.0 to 4.0 cm. Not to become an obstacle forbees entering or leaving the chambers, the openings should be close tothe floor members and are positioned up to about 0.5 cm above said floormembers, with diameters ranging from the width of the chambers down toabout 1.0 cm.

The two chambers as described herein could be independent and separatechambers, but preferably taken together form a single housing such asfor example provided in the different embodiments herein below. As suchthe housing can be used in or outside the hive. In the latter case theentrance(s) of the dispenser chamber and the exit(s) of the secondchamber will comprise means to communicate with the exit(s), respectiveentrance(s) of the hive.

The means to communicate with the hive entrance or exit are typicallyselected from flexible tubes, rigid tubes, swing doors or conical tubes.

This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Additionally,throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

EXAMPLES

In all of the following examples different embodiments of the beehivedisseminators are presented and tested for their efficacy indistributing microbiological control agents (MCA) using the bumblebeeBombus terrestris.

As provided in more detail in the examples hereinafter, efficacy of thebeehive disseminators, i.e. dispensers was assessed by;

-   -   determining the loading of the bumblebees with MCA, by scoring        the CFU that could be recovered from the bees;    -   determining the impact of the dispenser on foraging intensity,        by counting in and out frequency for 30 minutes at different        time points during the day; and    -   determining the amount of MCA dispersed in the flowers when        tested under practical conditions in a closed greenhouse. The        amount of MCA dispersed in the flowers was scored as the CFU        that could be recovered thereof.

Example 1. Funnel-Dispenser

In all of the experiments with the F-dispenser the MCA consists ofseeds/propagules of the Fungi strains Trichoderma atroviride andHypocrea parapilulifera at a minimum concentration of 1,000,000 CFU(Colony Forming Units)/gram. This MCA is for example available under theCommercial product name BINAB® T Vector (Bio-Innovation AB, Sweden).However, any control agent, which does not interfere with the bees toperform normally, could be used.

Funnel-Dispenser Design

The Funnel-dispenser (F-dispenser) has the following characteristics(FIGS. 1A-1D):

-   -   It is a two-way dispenser with a trapezium basis, made of 3 mm        thick PVC, with a separated exit and entrance compartment ((2)        and (1), respectively)    -   The trapezium basis has a width of 7.2 cm to 18 cm    -   Compartment (2), is filled with the MCA, is 5.3 cm high, from        about 7.0 to 14.0 cm wide and from about 20-30 cm long. This        compartment contains one opening (4) that is connected with the        nest via a short plastic tube with a bumblebee-in-closer and 3        exit openings (5), with a bumblebee-in-closer.        Bumblebee-in-closers are used to prevent that bumblebees will        return to the nest via the wrong opening    -   The exit openings are placed as closely as possible to the        entrance opening, since it was observed that bumblebees leaving        the dispenser via an exit opening located far from the entrance        opening spend more time to find the correct entrance opening    -   Compartment (1) (Length about 20-30 cm×Width about 3.0 cm×Height        5.3 cm) has two openings, one is connected with the nest (3) via        a short plastic tube, with a bumblebee in-closer at the inside        of the hive to prevent bumblebees from exiting the nest via the        wrong opening and the other is the entrance opening. The latter        opening (6) is marked at the outside with a blue color to        attract the bumblebees for going in    -   To avoid movement of the powder as a consequence of bumblebee        movement, a mesh is put on the bottom of the dispenser.

Funnel-Dispenser Efficiency for Bumblebee Loading

To determine the optimal minimal length of the Funnel-dispenser, toobtain an optimal loading of the bumblebee body, we drew lines on thedispenser (of 25 cm in length) at 5, 10, 15, 20 and 25 cm. The dispenserwas filled with BINAB® T Vector. Bumblebees of the same age and sizewere kept individually in a falcon tube covered with aluminum foil fordarkness. Then the falcon tube was connected with the dispenser, and thebumblebee workers were allowed to walk through the dispenser. Theworkers were captured at different distances after 5, 10, 15, 20 or 25cm of walking through the dispenser under complete darkness (red light)to avoid disturbance of the worker walking behavior by the presence oflight. After capturing, bees were killed, and the amount of CFU presenton their body was analyzed. Thereto for each distance, 10 bumblebeeswere shaken in a physiological solution at 200 rpm during 60 minutes.The amount of CFU was analyzed by plating out a series of dilutions ofthe obtained solution on selective media and for each dilution 4 plateswere used. Petriplates were incubated at room temperature and after 36hrs individual colonies were scored. The experiment was repeated threetimes.

As shown in FIG. 2, the best loading was obtained when the bees walkedfor 20 or 25 cm through the dispenser. The data suggest that the loadingreached its maximum/saturated loading with 20 and 25 cm of walkingthrough the dispenser. At lower distances a significantly (p<0.05) loweramount of CFU was recovered.

Furthermore, the efficiency of the F-dispenser for bumblebee loading wascompared to that of the previously described SSP-dispenser. Thereto anF-dispenser, in which the compartment filed with the MCA was 25 cm long,was used. The F-dispenser appeared to be much more effective inbumblebee loading, as the mean CFU/bee (433413±62780) was 10 timeshigher than for the SSP-dispenser (49421±3499). The mean body surfacearea of the bees used in both dispensers was comparable: 362.8±19.6 mm²for the SSP-dispenser and 356.7±59.0 mm², indicating that the differencein CFU/bee is due to a higher efficiency of the R-dispenser in bumblebeeloading.

Effect of Funnel-Dispenser on Foraging of the Bumblebees

The purpose of this experiment was to test whether attaching theFunnel-dispenser in front of a bumblebee hive, affects the foragingactivity of bumblebees in five different nests. To determine the periodwhen bees are most active, bumblebee activity (in/out of the nests) wascounted during 30-minute intervals between 7 AM-9 AM, 11 AM-1 PM, 4 PM-6PM and 7 PM-9 PM. Although there were no significant differences inbumblebee activity between the different time points, the highestactivity was observed during the 7 AM-9 AM and 11 AM-1 PM intervals(data not shown). Therefore, it was decided for the further analysis towork with 7 AM and 11 AM.

Next, bumblebee activity was determined at the time points 7 AM and 11AM in hives with and without an empty Funnel-dispenser, placed in frontof the hive. Measurement was done the day after placement of thedispenser. As shown in FIG. 5, although bumblebee activity was slightlylower in nests with an F-dispenser, this effect was not significant,indicating that the F-dispenser does not significantly affect bumblebeeactivity. The next morning (7-9 AM), the dispenser was loaded withBINAB® T Vector powder and the effect on bumblebee activity was measuredat 11 AM and 24 hours later, again at 11 AM (FIG. 3). Interestingly,bumblebee activity was significantly higher, 1 hr after loading of thedispenser as compared with an empty dispenser (FIG. 3), even beingcomparable to the bumblebee activity without the F-dispenser at 11 AM.Furthermore, a comparable level of activity was also observed 24 hoursafter loading of the F-dispenser.

Evaluation of the Funnel-Dispenser in a Closed Greenhouse

To evaluate the efficiency of the F-dispenser, we performed anexperiment under practical conditions in a closed greenhouse (controlledtemperature and humidity) with strawberries. In both the F-dispenser(0.08 g/cm²) as well as in a modified SSP dispenser (0.13 g/cm²)(separated entrance and exit openings) the BINAB® T Vector was used asto be dispersed agent. At weekly basis the powder level in thedispensers was scored and if necessary refilled. In total threegreenhouses were used: one for each dispenser and one control greenhousewhere no MCA was used. The temperature and relative humidity werefollowed with data loggers.

To determine how far bumblebee workers forage to inoculate flowers withMCAs, we marked on the ground floor of each greenhouse three circleswith the bumblebee nest in the centre and with a diameter of 8 m (zoneA), 18 m (zone B) and >18 m (zone C=the rest of the greenhouse). In eachzone, 20 flowers were collected and put individually in a falcon tube.This was done on a weekly base during 3 weeks. Per falcon tube 50 ml ofphysiological solution was added to the flowers, and these were shakenat 120 rpm during 60 min. A series of dilutions (1/1, 1/5,1/10-1/100,000) was prepared with the obtained solution and plated outon TSM (William et al., Applied and Environmental Microbiology, 2003,69, pg. 4190-4191). For each concentration 4 plates were used. After 36hrs of incubation at room temperatures, the numbers of colonies werescored.

Interestingly, more CFUs were scored per zone with use of theFunnel-dispenser compared to the modified SSP-system (FIG. 4). Betweenthe three zones A, B and C, there were no significant differences: equalamounts of CFU were recovered in the flowers.

In summary, our results clearly indicate that the F-dispenser does notsignificantly affect bumblebee activity and that it scores better in MCAloading on the bumblebees and in MCA deposition in flowers, compared tothe SSP dispenser.

Example 2. Rectangular-Dispenser

In the experiments with the R-dispenser the MCA consists of

-   -   seeds/propagules of the Fungi strains Trichoderma atroviride and        Hypocrea parapilulifera at a minimum concentration of 1,000,000        CFU (Colony Forming Units)/gram. This MCA is for example        available under the Commercial product name BINAB® T Vector        (Bio-Innovation AB, Sweden), or    -   seeds/propagules of the Fungi strain Gliocladium catenulatum        J1446 at a minimum concentration of 10,000,000 CFU (Colony        Forming Units)/gram. This MCA is for example available under the        Commercial product name Prestop Mix® (Verdera Oy, Finland).

However, any control agent, which does not interfere with the bees toperform normally, could be used.

Rectangular-Dispenser Design

The Rectangular-dispenser (R-dispenser) has the followingcharacteristics (FIGS. 5A-5D):

-   -   It is a two-way dispenser with a rectangular basis, made of 3 mm        thick PVC, with a separated exit and entrance compartment ((2)        and (1), respectively)    -   The rectangular basis has a width ranging of about 2.5 to about        15.0 cm, preferentially a width of about 10.0 cm.    -   Compartment (2), is filled with the MCA, is about 4.0 cm high,        5.0 cm wide and 20-25 cm long. This compartment contains one        opening (4) that is in direct contact with the nest and 2 exit        openings (5), with a bumblebee-in-closer. Bumblebee-in-closers        are used to prevent that bumblebees will return to the nest via        the wrong opening    -   The exit openings are placed as closely as possible to the        entrance opening, since it was observed that bumblebees leaving        the dispenser via an exit opening located far from the entrance        opening spend more time to find the correct entrance opening    -   Compartment (1) (Length about 8.0 cm×Width about 2.5 cm×Height        4.0 cm) has two openings, one is in direct communication with        the nest (3) and comprises a bumblebee in-closer, to prevent        bumblebees from exiting the nest via the wrong opening and the        other is in communication with the entrance opening (6) of the        hive through a short plastic tube. The latter opening is marked        at the outside with a blue color to attract the bees for going        in.    -   To avoid movement of the powder, as a consequence of bumblebee        movement, a mesh is put on the bottom of the dispenser.

R-Dispenser Efficiency for Bumblebee Loading

The efficiency of the R-dispenser for bumblebee loading wasinvestigated. Hereto the dispenser, filled with Prestop Mix® (0.11g/cm²), was connected to a bumblebee nest with two grey plastic tubes.In this nest, pollen was removed one day before connection, to stimulatebees to leave the dispenser and search for food. Then a falcon tube wasconnected in an angle of 45 degrees on the entrance/exit opening tocapture the worker bees that left the dispenser. In total 10 bumblebeeswere captured and killed. Thereafter the bumblebees were shaken in aphysiological solution at 200 rpm during 60 minutes. The amount of CFUwas analyzed by plating out a series of dilutions of the obtainedsolution on selective media (PDA) and for each dilution 4 plates wereused. The Petri dishes were incubated at room temperature and after 36hrs individual colonies were scored. The numbers of CFU recovered oneach body were calculated in respect to the surface area. The experimentwas repeated twice.

The mean CFU load per bumblebee worker was 1083150±3700 CFU and the meanbody surface per bumblebee worker was 361.0±18.0 mm². These dataindicate that the R-dispenser is even about 3,5 times more efficient forbumblebee loading compared to the F-dispenser with a travel length of 25cm (see FIG. 2).

Evaluation of the Effect of the R-Dispenser on Foraging Activity in aClosed Greenhouse

The purpose of this experiment was to compare the effect of theR-dispenser and the SSP-dispenser, both unloaded, on the foragingactivity of bees in a sweet pepper greenhouse. For each dispenser type,3 bumblebee hives were used, each consisting of one queen and 50workers. The control group consisted of regular bumblebee hives. At dayzero the two dispensers were connected to (for SSP-dispenser) or in (forR-dispenser) the regular hives. After 3 days, countings were done at 9AM and 4 PM. Countings were repeated at day 4 and day 5 at 9 AM and 4PM. Data were analyzed for normal distribution with a Kolmogorov-Smirnovtest.

The results followed a normal distribution. The foraging activity wasnot significantly different between 9 AM and 4 PM (data not shown). Themean foraging activity per 30 minutes for each dispenser type is shownin FIG. 6. These results indicate that the foraging activity per30-minute intervals was significantly (p<0.05) different between theR-dispenser and the SSP-dispenser. Moreover, no significant effect wasseen on the mean foraging intensity per 30 minutes between the controlsand the nests containing an R-dispenser.

Evaluation of the Effect of Loading the R-Dispenser on Foraging Activity

The purpose of this experiment was to test whether loading theR-dispenser, affects the foraging activity of bees. Thereto 10 nestscontaining an R-dispenser were used. At the evening of the next day (10PM) the R-dispensers in five nests were loaded with 10 g Binab® T-vectorand 5 stayed empty. The next morning counting was done at 7 AM, 11 AMand 4 PM to determine the effect of a dispenser loaded with powder andwithout powder. Any reduction in activity was attributed to the effectof the powder. Data were analyzed for normal distribution with aKolmogorov-Smirnov test. Then significance between the nests wasdetermined with an independent sample t-test.

The obtained results followed a normal distribution. We evaluated whatthe impact was of a loaded dispenser on the foraging intensity. For thefive nests with an empty R-dispenser, the response was not significantlydifferent at 7 AM, 11 AM and 4 PM as compared with the five nestscontaining a loaded R-dispenser (FIG. 7).

In summary, our results clearly indicate that the R-dispenser does notsignificantly affect bumblebee activity and that it scores very good inMCA loading on the bumblebees.

Example 3. Evaluation of the Effect of the Superimposed (S)-Dispenser onForaging Activity in Outside Conditions

The purpose of this experiment was to assess the effect of the presenceof the filled S-dispenser, on the foraging activity of bees in outsideconditions. Also, a modified S-dispenser was tested. This, called ‘3valves S-dispenser’ differed from the S-dispenser by featuring a thirdvalve between the dispenser chamber and the nest (see FIG. 10), topreclude loaded bees to return to the nest. Each dispenser type wastested in three replicates, each containing one queen and about 50workers. The control group consisted of 3 regular bumblebee hives. Thehives were placed in a row outside alternatingly. At day zero thedispensers were mounted in regular hives and filled with Maïzena tomimic the presence of a powder formulation. After 5 days, the dispenserswere refilled and countings of 30 minutes were done at 10 AM and 2:30PM. Countings were repeated at day 5, 7 and 9 at 10 AM and 2:30 PM.Averages and standard errors were calculated.

The mean foraging activity per 30 minutes for each dispenser type isshown in (FIG. 11). The foraging activity per 30-minute intervals wasnot significantly different between the two dispenser types and thestandard hive. Moreover, effect of the third valve added to the 3 valvesS-dispenser.

In summary, our results clearly indicate that the S-dispenser does notsignificantly affect bumblebee activity.

1-29. (canceled)
 30. A beehive comprising a disseminator device and atop grid, the disseminator device being an integrated part of the topgrid and comprising a dispenser chamber that holds a substance to bedisseminated, the dispenser chamber comprising an entrance and an exit.31. The beehive of claim 30, wherein the top member of the dispenserchamber consists of the covering grid of the hive.
 32. The beehive ofclaim 30, wherein the dispenser chamber comprises a top perforationthrough which the dispenser chamber is fillable with the substance. 33.The beehive of claim 30, further comprising a hinged or removable lidconnected to the top grid, through which the dispenser chamber isfillable with the substance.
 34. The beehive of claim 30, wherein theentrance of the dispenser chamber is closable.
 35. The beehive of claim30, further comprising means for immobilizing the substance to bedisseminated at the floor member of the dispenser chamber.
 36. Thebeehive of claim 30, further comprising a unidirectional entrance forbees entering the hive.
 37. The beehive of claim 30, further comprisinga unidirectional exit pathway for bees leaving the hive.
 38. The beehiveof claim 1, further comprising bees.
 39. The beehive of claim 38,wherein the bees are honeybees or bumblebees.
 40. The beehive of claim39, wherein the bees are bumblebees.
 41. A method for distributing abiological control agent, the method comprising: providing a beehiveaccording to claim 30; filling the dispenser chamber with a biologicalcontrol agent; and allowing bees to pass through the dispenser chamber,whereby the biological control agent is distributed when the bees forageoutside of the beehive.
 42. The method of claim 41, wherein the bees arehoneybees or bumblebees.